Impact of arabinoxylan-enriched wheat flour on sourdough production

As a staple food, bread is one of the most consumed food products in the world. The revival of sourdough breadmaking has its roots in not only the perception of its more natural and traditional character compared to mainstream industrial bread production, but also its positive impact on the aroma, taste, shelf-life, and nutritionally-related aspects of the final baked products. A sourdough is the result of a flour-water mixture that is subjected to an acidifying fermentation process by a consortium involving lactic acid bacteria (LAB), yeasts, and occasionally acetic acid bacteria, which contribute to flavour formation and dough leavening. These microbial consortia can either develop spontaneously upon periodic refreshments, as the result of starter culture addition, or as a combination of both techniques.

Regarding their nutritional composition, wheat bakery products represent a substantial percentage of the daily caloric and dietary fibre intake in European diets. Efforts to increase the dietary fibre consumption may benefit from the availability of wheat varieties with an increased fibre content in the endosperm, as refined wheat flours are still favoured over their wholemeal counterparts. Besides nutritional and health outcomes, arabinoxylan (AX) fibres play a significant role in breadmaking technology because they can impact the gluten network formation, depending on their structural features. Depending on their extractability in water, AX can be water-extractable (WE-AX) or water-unextractable (WU-AX). Whereas WE-AX have a neutral or positive impact on the dough rheology and specific bread volume, WU-AX have a negative impact.

During the present PhD study, the impact of AX-enriched wheat flour on the flour fermentation process for sourdough production was evaluated. To this end, first a downscaling of the backslopped sourdough production process was carried out with triticale flour, a hybrid of wheat and rye and an under-utilized cereal crop for sourdough production. This was followed by the production of backslopped sourdoughs with wheat flour blends that possessed an increased content of AX. These spontaneously fermented sourdoughs were assessed following a multiphasic approach, involving a culture-dependent (selective plating and incubation, followed by molecular identification of picked colonies) and culture-independent (metagenetics of whole-community DNA) microbial community analysis, as well as the determination of the substrates consumed, and the metabolites produced (metabolite target analysis) during flour fermentation. The resulting sourdoughs were microbiologically diverse, involving the presence of both homo- and heterofermentative LAB species, such as Limosilactobacillus fermentum, Pediococcus pentosaceus, Lactococcus lactis, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and Levilactobacillus brevis. The scale of the sourdough production played a role in shaping the microbial consortia, whereby a smaller scale resulted in both a lower microbial diversity and a faster establishment of a stable consortium than for the sourdoughs produced at larger scale. An increase in the content of AX in wheat flour did not result in a specific or atypical consortium of LAB and/or yeasts, but fermentation of such wheat flour blends resulted in a consistent increase of the WE-AX fraction, which could be ascribed to the acidification taking place during flour fermentation.

Building on the knowledge obtained during the backslopped sourdough productions with high-AX wheat flour, the potential for AX degradation by sourdough LAB was assessed. First, an in silico genome mining was carried out, revealing that genes encoding AX-degrading enzymes were present in the genomes of LAB strains with sourdough origin available in the IMDO-VUB culture collection. Next, following a rational selection, four LAB strains were further investigated, namely, Liml.
fermentum IMDO 130101, Lactococcus lactis IMDO WA12L8, Companilactobacillus crustorum LMG 23699, and Companilactobacillus paralimentarius IMDO BBRM18. Starter culture-initiated sourdoughs were produced with these strains using a high-AX wheat flour blend to assess the impact of flour fermentation on the structural characteristics of WE-AX. The use of long-read sequencing technologies, targeting the 16S rRNA gene for an amplicon sequence variant (ASV) analysis of the bacterial communities, confirmed the prevalence of the starter culture strains inoculated in the flour-water mixture for the sourdough productions.

Next, the structural features of the WE-AX present in the high-AX wheat flour used for the sourdough productions were studied as a function of the fermentation time, involving the use of advanced nuclear magnetic resonance (NMR) techniques such as diffusion-ordered spectroscopy (DOSY)-NMR. As fermentation time progressed, the WE-AX became smaller in size, but their average composition, i.e., the average arabinose-to-xylose substitutions did not vary. These results pointed to the absence of arabinofuranosidase activity and hinted toward the importance of acid-induced activation of endogenous endo-xylanases present in the wheat flour. The reduction of the AX size may have implications for the digestibility of these fibre compounds in the gut, as well as have an impact on
breadmaking technology. More specifically, the use of sourdough and uninhibited commercial xylanases during breadmaking resulted in a too extensive AX solubilization, having a negative impact on the specific bread volume.

Concerning the production of microbial metabolites, new insights were obtained. As an example, the heterofermentative Liml. fermentum IMDO 130101 was recurrently correlated with the simultaneous production of lactic acid, ethanol, mannitol, acetic acid, glycerol, erythritol, and ethyl lactate. Of particular notice was the biosynthesis of erythritol. This sugar alcohol was produced in low concentrations and favoured to the detriment of glycerol during model fermentations in wheat sourdough simulation media, but more research is needed to identify and characterize the enzymes responsible for this production. Further, the homofermentative Lc. lactis IMDO WA12L8 was linked to the production of buttery flavour compounds, such as acetoin, diacetyl, and 2,3-butanediol.

In conclusion, the research presented in this PhD thesis provided insights into the establishment of diverse microbial consortia during backslopped sourdough productions carried out with triticale flour and high-AX wheat flour. Relevant links between microorganisms and the metabolites produced were drawn, which were confirmed upon starter culture-initiated sourdough productions, both in wheat sourdoughs and wheat sourdough simulation medium. Finally, it was shown that high-AX wheat flour fermentation resulted in an increase in the content of WE-AX, regardless of the prevailing LAB species, and furthermore, that those WE-AX were of a reduced molecular size.